Neuroprotective function of cellular prion protein in a mouse model of amyotrophic lateral sclerosis

Abstract

Transgenic mice expressing human mutated superoxide dismutase 1 (SOD1) linked to familial forms of amyotrophic lateral sclerosis are frequently used as a disease model. We used the SOD1G93A mouse in a cross-breeding strategy to study the function of physiological prion protein (Prp). SOD1G93APrp-/- mice exhibited a significantly reduced life span, and an earlier onset and accelerated progression of disease, as compared with SOD1G93APrp+/+ mice. Additionally, during disease progression, SOD1G93APrp-/- mice showed impaired rotarod performance, lower body weight, and reduced muscle strength. Histologically, SOD1G93APrp-/- mice showed reduced numbers of spinal cord motor neurons and extended areas occupied by large vacuoles early in the course of the disease. Analysis of spinal cord homogenates revealed no differences in SOD1 activity. Using an unbiased proteomic approach, a marked reduction of glial fibrillary acidic protein and enhanced levels of collapsing response mediator protein 2 and creatine kinase were detected in SOD1G93APrp-/- versus SOD1G93A mice. In the course of disease, Bcl-2 decreases, nuclear factor-kappaB increases, and Akt is activated, but these changes were largely unaffected by Prp expression. Exclusively in double-transgenic mice, we detected a significant increase in extracellular signal-regulated kinase 2 activation at clinical onset. We propose that Prp has a beneficial role in the SOD1G93A amyotrophic lateral sclerosis mouse model by influencing neuronal and/or glial factors involved in antioxidative defense, rather than anti-apoptotic signaling.

Figure 1

Diagrams showing data on disease onset, progression, and survival of SOD1^G93A transgenic mice (n = 12 males and females, respectively) and SOD1^G93APrp−/− mice (n = 10 males, n = 12 females). Kaplan Meier survival curves of male (A) and female (B) SOD1^G93A transgenic mice dependent on Prp expression. The log rank P values are shown. C: On the left, the diagram shows Prp dependent age of SOD1^G93A male and female mice when developing first clinical disease signs (gait impairment) and when developing first pareses. On the right, the difference in the duration of early disease phase (ie, from peak of body weight curve until the time when mice lost 10% body weight) and of the late disease phase (ie, 10% body weight loss until terminal disease stage) is illustrated. Data are expressed as mean ± SD. Analyses by Mann-Whitney test revealed significant differences in age at disease onset in males (P = 0.007), onset of pareses in males (P < 0.001) and females (P = 0.019), and duration of early disease phase (P = 0.024). Body weight (D), rotarod performance (E), and GRIP strength (F) of the control mouse groups (wild-type and Prp−/−), and of Prp expressing and Prp deficient SOD1^G93A transgenic mice. The difference in body weight of SOD1^G93A and SOD1^G93APrp−/− group was significant from day 140 (P = 0.018) until end of experiment, as indicated by asterisks (d147 P = 0.005, d154 P = 0.034, d161 P = 0.022). GRIP strength and rotarod performance were determined tree-times for each tested animal per week. Plotted are mean ± standard deviations. Significant differences in the SOD1^G93A and the SOD1^G93APrp−/− group are indicated by asterisks.

Figure 2

Representative spinal cord slices and motor neuron counts from wild-type, Prp−/−, SOD1^G93A and SOD1^G93APrp−/− mice. A: The upper panel shows tissue from mice expressing wild-type SOD1. Arrows depict healthy motor neurons. In SOD1^G93A transgenic mice at disease onset (middle panel) and at final disease stage (lower panel), the typical degeneration of motor neurons (arrows from the right) and vacuolization (arrows from the left) can be seen. Pictures were taken from H&E-stained 7-μm sections of formalin-fixed tissue scale bars = 50 μm. B: Overviews of cervical spinal cord regions of male mice at time of disease onset. In the Prp−/− tissue (top) no vacuolization can be seen, as is the case in SOD1^G93A anterior horn (middle) and, to a greater extent, in SOD1^G93APrp−/− tissue (bottom). Scale bars = 200 μm. C: Number of healthy motor neurons counted in lumbar, thoracic, and cervical spinal cord regions of mice at disease onset (SOD1^G93An = 3, SOD1^G93APrp−/− n = 2) for each genotype), of mice at final disease stage (SOD1^G93A and SOD1^G93APrp−/−, n = 3, respectively) and of respective controls (wild-type and Prp−/− n = 3, respectively). Seven sections 49 μm apart (or in case of cryo-conserved tissue, 84 μm apart) were evaluated for each animal and region and the mean was calculated. In the diagram mean values ± standard deviations of the individual mice of a certain genotype are given. The differences between SOD1^G93A and SOD1^G93APrp are significant (P > 0.05) with exception of the cervical region at final disease stage. P values for disease onset were P = 0.0067 (lumbar), P = 0.0039 (thoracic) and P = 0.0023 (cervical) and for terminal disease stage P = 0.0272 (lumbar) and P = 0.0002 (thoracic). No differences in motor neuron numbers were detected comparing wild-type with Prp−/−.

Figure 3

SOD activities determined in lysates of spinal cord homogenates of male mice at final disease stage. Animals with the genotypes Prp+/+ (n = 2), Prp−/− (n = 2), SOD1^G93A (n = 4) and SOD1^G93APrp−/− (n = 4) are compared. The activities of total SOD, of MnSOD in the presence of cyanide to block Cu/ZnSOD, and of Cu/ZnSOD in the presence of ethanol/chloroform to block MnSOD, are shown.

Figure 4

A: 2D-DIGE analysis of whole brain homogenates of two individual SOD1^G93A male mice either expressing Prp (Prp+/+, red) or Prp knockout (Prp−/−, green). Internal standard is shown in yellow and represents pooled total brain homogenate protein of three SOD1^G93APrp+/+ and three SOD1^G93APrp−/− male mice. The inserts show spots with significantly reduced abundance in SOD1^G93APrp−/− mice later identified as GFAP. B: Quantification of GFAP immunoblots of spinal cord homogenates of terminally ill female mice (SOD1^G93An = 4, SOD1^G93APrp−/− n = 4, wild-type n = 10, Prp−/− n = 6) and (C) of male mice at disease onset (SOD1^G93An = 7, SOD1^G93APrp−/− n = 4, wild-type n = 7, Prp−/− n = 7). Underlying data were normalized against tubulin bands detected on the same blot after stripping of the membranes. Above the diagrams, representative blots for both disease stages and each genotype are shown. Asterisks in (B) and (C) indicate significant differences between two groups. (D-F) GFAP immunostaining of cervical spinal cord sections (7 μm) of a wild-type (D), a SOD1^G93APrp+/+ (E), and of a SOD1^G93APrp−/− mouse (F). Astrocytosis can be seen in both SOD1 transgenic mouse tissues. In areas undergoing massive vacuolization GFAP staining seems to be less intense in the Prp knockout (F, left panel). Focusing on areas with less vacuoles (F, right panel) there is no clear difference in density of GFAP staining or number of astrocyte cell bodies between SOD1^G93A and SOD1^G93APrp−/− tissues.

Figure 5

Representative immunoblots of spinal cord homogenates of male mice at disease onset, defined as time first clinical symptoms are visible, and of female mice at terminal disease stage. Data are expressed as mean ± SD. Significant changes are indicated by asterisks. A: ERK1/2 expression at disease onset (n = 5 wild-type, n = 3 Prp−/−, n = 5 SOD1^G93A, n = 4 SOD1^G93APrp−/− mice), and (B) of terminal disease (n = 4 for each group). Diagrams show the ratio of band intensities of phosphorylated to unphosphorylated ERK. The pERK2/ERK2 ratio is significantly increased in SOD1^G93A mice when Prp is knocked out (P = 0.0159). Relative Bcl-2 expression levels at disease onset (n = 4 wild-type, n = 6 Prp−/−, n = 3 SOD1^G93A, n = 4 SOD1^G93APrp−/−)(C) and at the end of disease (n = 4 each genotype) (D). In the terminal disease stage, Bcl-2 was significantly decreased in both SOD1^G93A transgenic groups without differences due to Prp expression. E: Expression levels of Akt and pAkt and the ratio pAkt/Akt in wild-type (n = 3), Prp−/− (n = 4), SOD1^G93A (n = 9), and SOD1^G93APrp−/− (n = 6) male mice at disease onset. At the top, representative Akt and pAkt blots for each genotype are given as in (F), which illustrates the results obtained analyzing female mice of the terminal disease stage. pAkt is increased significantly in ALS mice, independently of Prp genotype (wild-type versus SOD1^G93AP = 0.0153 and Prp−/− vs SOD1^G93APrp−/− P = 0.0364). The ratio pAkt/Akt is significantly different only in the Prp−/− versus SOD1^G93APrp−/− group (P = 0.0141). G and H: Representative NF-κB immunoblots and quantification of expression levels at disease onset (G) and at final disease stage (H), respectively. Analyzed were n = 4 wild-type, n = 3 Prp−/−, n = 6 SOD1^G93A, and n = 6 SOD1^G93APrp−/− male mice, and n = 3 wild-type, n = 3 Prp−/−, n = 7 SOD1^G93A, and n = 6 SOD1^G93APrp−/− female mice. At final disease, a significant induction of NF-κB could be observed in the Prp+/and Prp−/− group (wild-type versus SOD1^G93AP = 0.0476 and Prp−/− versus SOD1^G93APrp−/− P = 0.0083).